The present application is directed primarily at understanding the transcriptional regulation of histone variant H1t. H1t is a testis-specific linker histone that is synthesized only in mid to late pachytene spermatocytes. H1t may help to relax chromatin structure to facilitate both the dramatic changes in gene expression that accompany spermatogenesis as well as the histone to transition protein/protamine changeover that occurs in condensing spermatids. H1t is an excellent example of a spermatocyte-specific gene. Understanding its transcriptional control is expected to help understand the ordered changes in transcriptional factors that control spermatogenesis. This knowledge will aid in the molecular diagnosis of spermatogenic arrest in infertile males and point to possible therapeutic strategies. The H1t promoter shares many functional DNA motifs with standard H1 histones, but these regulatory elements must be subservient to additional elements that impart strict cell-type specificity. Prior studies identified two such DNA motifs: a pair of palindromic sites, which are proposed to promote spermatocyte activation, as well as a strong silencer element downstream of the TATA box, which suppresses somatic expression. We will demonstrate the critical importance of these elements, using transgenic mice to analyze the effects of targeted mutations in each motif on H1t expression. We will also use oligonucleotide affinity procedures to purify small amounts of the nuclear proteins that bind these two sites. The proteins will be identified by mass spectrometry and database searches, and this information will be used to obtain full-length cDNA clones and prepare specific antisera. The functional importance of the palindrome binding factor will be demonstrated by creating a targeted gene disruption in transgenic mice (gene knockout). The in vivo relevance of the protein to promoter activation will be shown by ChIP assays, and additional studies will be done to understand the function of this transcriptional transactivator.